Project Summary Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. How the collective invasion pack operates, communicates, and navigates as a single cohesive unit remains unclear. To address this, we published on an image-guided genomics platform to isolate any living cell(s) within a collective invasion pack, and expand the population for genomic and molecular analysis, a technique termed Spatiotemporal Cellular & Genomic Analysis (SaGA). We used SaGA to dissect the molecular, epigenetic, and genomic profiles of leader and follower cells invading as a hierarchical cohesive unit. To determine how epigenetic reprogramming drives this phenotypic heterogeneity, we deconstructed the collective invasion pack using SaGA, then integrated genome-wide promoter methylation and transcriptome data to define differentially methylated regions within the leader and follower phenotypes. We observe global epigenomic re-wiring in leader cells supporting an epigenetic basis for the phenotypic heterogeneity within the collective invasion pack. We then identified Myo10 (myosinX) as a top differentially methylated and expressed gene, where the leader cell promoter is hypomethylated, and leaders in several lung cancer lines overexpress Myo10. Myo10 is a canonical modulator of filopodia elongation and we show it drives filopodia elongation, collective invasion, leader cell-driven fibronectin micropatterning (fibrillogenesis), and is transcriptionally activated by Jag1/Notch. We will use this information to test a mechanistic model with the overarching hypothesis that Myo10 activation via promoter hypomethylation in leader cells drives filopodia-based micropatterning of fibronectin to create a leader cell-driven collective invasion path. We propose that this leads to an invasive advantage for lung cancer cells resulting in metastatic disease. In Aim 1 we test the model that Myo10 hypomethylation in leaders allows for Jag1/Notch1- driven transcriptional activation, driving filopodia elongation, and fibronectin micropatterning. In Aim 2 we test how this collective invasion pathway impacts metastasis using in vivo metastasis models and the first patient- derived leader cells. Throughout, we leverage unique resources developed here including SaGA-derived cell lines, ex vivo imaging, and patient-derived lung cancer leader cells. We speculate that these data will provide mechanistic insight into collective invasion and translational value towards understanding lung cancer patient leader cell biology.